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Development of Dynamic DNA Probes for High-Content in situ Proteomic Analyses

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dc.contributor.advisor Diehl, Michael R.
dc.creator Schweller, Ryan
dc.date.accessioned 2012-09-06T04:00:09Z
dc.date.accessioned 2012-09-06T04:00:13Z
dc.date.available 2012-09-06T04:00:09Z
dc.date.created 2012-05
dc.date.issued 2012-09-05
dc.date.submitted May 2012
dc.identifier.uri http://hdl.handle.net/1911/64649
dc.description.abstract Dynamic DNA complexes are able to undergo multiple hybridization and dissociation events through a process called strand displacement. This unique property has facilitated the creation of programmable molecular detection systems and chemical logic gates encoded by nucleotide sequence. This work examines whether the ability to selective exchange oligonucleotides among different thermodynamically-stable DNA complexes can be harnessed to create a new class of imaging probes that permit fluorescent reporters to be sequentially activated (“turned on”) and erased (“turned off”). Here, dynamic DNA complexes detect a specific DNA-conjugated antibody and undergo strand displacement to liberate a quencher strand and activate a fluorescent reporter. Subsequently, incubation with an erasing complex allows the fluorophore to be stripped from the target strand, quenched, and washed away. This simple capability therefore allows the same fluorescent dyes to be used multiple times to detect different markers within the same sample via sequential rounds of fluorescence imaging. We evaluated and optimized several DNA complex designs to function efficiently for in situ molecular analyses. We also applied our DNA probes to immunofluorescence imaging using DNA-conjugated antibodies and demonstrated the ability to at least double the number of detectable markers on a single sample. Finally, the probe complexes were reconfigured to act as AND-gates for the detection of co-localized proteins. Given the ability to visualize large numbers of cellular markers using dynamic DNA probe complexes, high-content proteomic analyses can be performed on a single sample, enhancing the power of fluorescence imaging techniques. Furthermore, dynamic DNA complexes offer new avenues to incorporate DNA-based computations and logic for in situ molecular imaging and analyses.
dc.format.mimetype application/pdf
dc.language.iso eng
dc.subject DNA Nanotechnology
Molecular Imaging
Strand Displacement
Nucleic Acids
Fluorescent Probes
Immunochemistry
dc.title Development of Dynamic DNA Probes for High-Content in situ Proteomic Analyses
dc.contributor.committeeMember Qutub, Amina A.
dc.contributor.committeeMember Farach-Carson, Mary C.
dc.date.updated 2012-09-06T04:00:14Z
dc.identifier.slug 123456789/ETD-2012-05-105
dc.type.genre thesis
dc.type.material text
thesis.degree.department Bioengineering
thesis.degree.discipline Bioengineering
thesis.degree.grantor Rice University
thesis.degree.level Doctoral
thesis.degree.name Doctor of Philosophy
dc.embargo.terms 2014-09-05T05:00:00Z
dc.embargo.lift 2014-09-05T05:00:00Z
dc.identifier.citation Schweller, Ryan. (2012) "Development of Dynamic DNA Probes for High-Content in situ Proteomic Analyses." Doctoral Thesis, Rice University. http://hdl.handle.net/1911/64649.

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